The incidence of hospital acquired infections has been increasing in recent years at an alarming rate which has caused great concern among the staffs of hospitals and especially those working in the laboratories. While many disinfection and sterilization techniques have been employed to alleviate this problem in different functional sections of the hospital, these techniques have not consistently provided a safe environment for the staff. Frequently, the disinfection and sterilization techniques which have been used have been employed after overt contamination has taken place from spilling, broken samples, etc. While these techniques have helped to reduce the incidence of laboratory acquired infections, they have not curtailed them. With the increasing incidence of contagious pathogens that can be transmitted by patient's specimens, especially blood and particularly such dangerous contaminants as the AIDS and hepatitis viruses, a new and safe technique for handling laboratory specimens is needed.
Various disinfectants and sterilizing agents have been employed with varying degrees of success, both in hospitals and other environments, Monoaldehydes such as formaldehyde have been used successfully as a disinfectant, however, dialdehydes, particularly glutaraldehyde, are preferred. Examples of glutaraldehyde-based disinfectants are a dilute sodium phenate-glutaraldehyde solution buffered to pH 7.4, an activated solution which contains 2.0% glutaraldehyde buffered to pH 7.5-8.0 and a disinfectant and sterilizing solution containing 2% glutaraldehyde at pH 7.0-7.5.
The extensive use of glutaraldehyde based compositions as an antiseptic and disinfectant has led to extensive of the compound and its activity. Glutaralde been classified as a chemosterilizer and has been Borick, J. of Pharm. Sciences, vol. 53, no. 10, Oct. 1964, as a chemical agent capable of destroying all forms of microbiological life including bacterial fungus spores, tubercle bacilli and viruses. The compound in fact been shown to be effective against a wide range of viruses even in the presence of high levels of organic matter which tend to destroy the potency of other disinfectants. The degree of biocidal activity observed in glutaraldehyde solutions is very much dependent on the pH of the solution as enhanced biocidal activity is found in alkaline solutions.
Boucher et al., Proc. West Pharmacal Soc. 16, pp. 282-288, 1973, postulated that the biocidal activity of gluaraldehyde is controlled by the distance between the aldehyde groups and their tendency to polymerize, thereby allowing free aldehyde groups to interact with the amino groups of the bacterial cell. This agrees with the findings of Rubbo et al., J. Appl. Bacteriol 30, pp.78-87, 1967, that antibacterial activity is due to the two aldehyde groups present on the molecule. After considering these results, Navarro and Monsan, Ann. Microbol 127B, pp.295-307, 1976, concluded that only structures containing two aldehyde groups allow formation of an aldol type polymer at an alkaline pH, and also produces a similar sterilizing effect at acid pHs on increasing concentrations. In other words, while the extent of polymerization is considerable at alkaline pHs, it is negligible in acid solutions unless the concentration is increased. On the other hand, acid solutions at pH3-4 of glutaraldehyde are considerably more stable than alkaline solutions.
The antimicrobal activity in any compound can not be viewed in isolation but must be described with reference to a number of factors including pH, temperature, organic matter present, and concentration. For glutaraldehyde, it has been common to use a 2% solution at room temperature and an alkaline pH of about 7.9. Unfortunately, alkaline solutions of glutaraldehyde are much less stable than acid solutions owning to the polymerization reactions already described, with a corresponding loss of antimicrobiol activity. A reduction in sporicidal activity of activated glutaraldehyde on storage has been observed in reports of Kelsey et al., J. Clin. Pathol. 27, pp.632-638, 1974, Thomas and Russell, J. Appl. Microbiol 28, pp.331-225, 1974b, Gorman and Scott, Int. J. Pharma 4, pp.57-65, 1979a. This reduction in sporicidal activity is directly related to a drop in concentration of the free aldehyde which appears to be essential for biological activity. Borick, Adv. Appl. Microbiol 10, pp. 291-312, 1968, has estimated that glutaraldehyde concentration actually falls from 2.1% at pH 8.5 to 1.3% at pH 7.4 over a period of twenty-eight days at ambient temperatures. Accordingly, it has generally been the practice to employ glutaraldehyde as a 2% solution to which an activator is added to bring the pH to approximately 8 at the time of use. Such a solution used at room temperature will, for example, disinfect within 10 minutes and sterilize within 10 hours. However, it has been recommended that this solution be discarded after 14 days because of the significant decrease in activity and free aldehyde concentration. This instability has lead to the development of more stable preparations formulated at lower pHs and some with other potentiators included to increase the otherwise low level of activity observed at lower pH.
The inevitable conditions of clinical use for disinfection and sterilization frequently mean that organic matter is present such as blood and pus. This organic matter can act either by protecting the microbial species from antimicrobial attack or by competing with the microbial cell for active sites on the disinfectant molecules, thus reducing the effective concentration of disinfectant substance. Accordingly, many otherwise effective disinfectants and sterilizing agents may become ineffective where organic material, such as blood, is contacted. Glutaraldehyde, however, has a high resistance to neutralization by organic matter. Borick et al., J. Pharm. Sci. 53, pp. 1273-1275, 1964, for example has reported that the presence of 20% blood serum did not appear to adversely effect the activity of glutaraldehyde while Synder and Cheatle, Am. J. Hosp. Pharm. 22, pp. 321-327, 1965, have reported that 1% whole blood did not effect glutaraldehyde activity.
One of the most important considerations in selecting a suitable disinfectant, in addition to its potency and sustained effectiveness as a disinfectant, is the toxicity of the composition to individuals coming in contact with it. Various studies have shown that glutaraldehyde, in moderate effective concentrations, is generally only slightly irritating to the skin, mucous membranes and eyes. Sato and Dobson, Arch. Dermatol 100, pp. 564-569, 1969, have found that 5% glutaraldehyde was only irritating if the epidermal barrier was not intact.
Aqueous solutions of glutaraldehyde have been used to treat hyperhydrosis and it has been used topically in the treatment of onychomycosis. Prevention of dental calculous formation and reduction of dental cavity formation in the mouth has been achieved by using oral compositions incorporating glutaraldehyde. In the cosmetic field, glutaraldehyde has been proposed for disinfection of production equipment and as a preservative. Glutaraldehyde has been used as a disinfectant for control of mastitis.
Accordingly, glutaraldehyde is now a generally accepted disinfectant and is found in a number of commercial preparations for disinfection and sterilization. Babb et al., J. Hosp. Infec. 1, pp. 63-75, 1980, for example, have compared nine glutaraldehyde products.
Glutaraldehyde has also been used extensively in various non-microbiological areas including the leather tanning industry and tissue fixation for electromicroscopy. In microbiological areas, glutaraldehyde has been employed principally as a liquid chemical sterilizing agent for medical and surgical material that cannot be sterilized by heat or irradiation. Compared with other disinfectants, glutaraldehyde has been found to be superior for disinfection of face masks, breathing tubes and other respiratory therapy equipment. Important advantages of glutaraldehyde as a chemosterilizer are: its activity in the presence of organic material, non-corrosive action towards metals, rubber, lenses and most materials, and lack of deleterious effect on cement and lenses of endoscopes. Further, glutaraldehyde has been recommended for decontamination of dental, surgical instruments and working surface where the hepatitis B surface antigen may be present as well as for the treatment of warts.
From the above mentioned studies, testing any biological specimen containing glutaraldehyde will not damage the instrument used in testing. Osterberg, Arch. Pharm. Chemi. Sci. Ed. 6, pp. 241-248, 1978, found that damage to leukocytes was apparent only above a 100 microg/ml. glutaraldehyde level. In addition, no erythrocyte damage occurred at the glutaraldehyde concentrations used.
The use of aldehydes in electron microscopy was extensively studied and it was found that many cytochemical reactions can be performed on tissue specimens after aldehyde fixation. Glutaraldehyde is effective in preserving both prokaryotes and eukaryotes, including fragile specimens such as marine invertebrates, embryos, diseased cells and fungi. Glutaraldehyde stabilized blood plasma with little shrinkage of blood clots (Chambers et al. 1968 Arch. Pathol. 85,18.). Tissue specimens can be left in this fixative for many hours without apparent deterioration. Presently, glutaraldehyde is the most efficient and reliable fixative for preservation of biological specimens for routine electron microscopy and the previously mentioned and available data indicate that proteins are not denaturated to any marked extent by fixation with glutaraldehyde (M. A. Hayat, Fixation for Electromicroscopy, Academic Press, 1981). Similarly, glutaraldehyde fixed-erythrocytes remain sensitive to the hemagglutination and hemagglutination inhibition tests for arbovirus antigens and antibodies (Wolff et al. [1977] J. Clin Microbiol. 6.55). Differential staining of viable and nonviable cells with alcian blue is maintained after fixation with glutaraldehyde (Yip and Auerperg, 1972, In Vitro 7, 323). From the above mentioned studies, glutaraldehyde will preserve the biological specimens without otherwise affecting the integrity of the specimen for future evaluation.
As set forth above, the handling of biological specimens such as blood after sampling, during storage and medical evaluation poses a particular hazard for those coming in contact with the specimens, especially where there is a possibility of AIDS (HTLV-III) or Hepatitis Virus being present. Despite the known effectiveness of disinfectants such as glutaraldehyde in destroying these viruses, their use has essentially been limited to the containers and equipment coming in contact with the fluid, and only after such contact has occurred and the fluid disposed of. What remains especially hazardous is the contaminated body fluids themselves, such as AIDS (HTLV-III) or Hepatitis infected blood, which are carriers of the infection from the time they are drawn from the donor. Accordingly, what is needed is a technique for destroying such viral contamination instantaneously when the sample is taken, but without effecting the specimens for further testing.